Plasminogen promotes cholesterol efflux by the ABCA1 pathway Nathalie Pamir, … , Godfrey S. Getz, Jay W. Heinecke JCI Insight. 2017;2(15):e92176. https://doi.org/10.1172/jci.insight.92176. Research Article Endocrinology Metabolism Using genetic and biochemical approaches, we investigated proteins that regulate macrophage cholesterol efflux capacity (CEC) and ABCA1-specific CEC (ABCA1 CEC), 2 functional assays that predict cardiovascular disease (CVD). Macrophage CEC and the concentration of HDL particles were markedly reduced in mice deficient in apolipoprotein A-I (APOA1) or apolipoprotein E (APOE) but not apolipoprotein A-IV (APOA4). ABCA1 CEC was markedly reduced in APOA1-deficient mice but was barely affected in mice deficient in APOE or APOA4. High-resolution size-exclusion chromatography of plasma produced 2 major peaks of ABCA1 CEC activity. The early-eluting peak, which coeluted with HDL, was markedly reduced in APOA1- or APOE-deficient mice. The late-eluting peak was modestly reduced in APOA1- deficient mice but little affected in APOE- or APOA4-deficient mice. Ion-exchange chromatography and shotgun proteomics suggested that plasminogen (PLG) accounted for a substantial fraction of the ABCA1 CEC activity in the peak not associated with HDL. Human PLG promoted cholesterol efflux by the ABCA1 pathway, and PLG-dependent efflux was inhibited by lipoprotein(a) [Lp(a)]. Our observations identify APOA1, APOE, and PLG as key determinants of CEC. Because PLG and Lp(a) associate with human CVD risk, interplay among the proteins might affect atherosclerosis by regulating cholesterol efflux from macrophages. Find the latest version: https://jci.me/92176/pdf RESEARCH ARTICLE Plasminogen promotes cholesterol efflux by the ABCA1 pathway Nathalie Pamir,1 Patrick M. Hutchins,1 Graziella E. Ronsein,1 Hao Wei,1 Chongren Tang,1 Riku Das,2 Tomas Vaisar,1 Edward Plow,2 Volker Schuster,3 Marlys L. Koschinsky,4Catherine A. Reardon,5 Richard Weinberg,6 David A. Dichek,1 Santica Marcovina,1 Godfrey S. Getz,7 and Jay W. Heinecke1 1Department of Medicine, University of Washington, Seattle, Washington, USA. 2Department of Molecular Cardiology, Cleveland Clinic, Cleveland, Ohio, USA. 3Hospital for Children and Adolescents, Medical Faculty of Leipzig University, Leipzig, Germany. 4Robarts Research Institute, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada. 5Ben May Research Institute, University of Chicago, Illinois, USA. 6Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA. 7Department of Pathology, University of Chicago, Chicago, Illinois, USA. Using genetic and biochemical approaches, we investigated proteins that regulate macrophage cholesterol efflux capacity (CEC) and ABCA1-specific CEC (ABCA1 CEC), 2 functional assays that predict cardiovascular disease (CVD). Macrophage CEC and the concentration of HDL particles were markedly reduced in mice deficient in apolipoprotein A-I (APOA1) or apolipoprotein E (APOE) but not apolipoprotein A-IV (APOA4). ABCA1 CEC was markedly reduced in APOA1-deficient mice but was barely affected in mice deficient in APOE or APOA4. High-resolution size-exclusion chromatography of plasma produced 2 major peaks of ABCA1 CEC activity. The early-eluting peak, which coeluted with HDL, was markedly reduced in APOA1- or APOE-deficient mice. The late- eluting peak was modestly reduced in APOA1-deficient mice but little affected in APOE- or APOA4- deficient mice. Ion-exchange chromatography and shotgun proteomics suggested that plasminogen (PLG) accounted for a substantial fraction of the ABCA1 CEC activity in the peak not associated with HDL. Human PLG promoted cholesterol efflux by the ABCA1 pathway, and PLG-dependent efflux was inhibited by lipoprotein(a) [Lp(a)]. Our observations identify APOA1, APOE, and PLG as key determinants of CEC. Because PLG and Lp(a) associate with human CVD risk, interplay among the proteins might affect atherosclerosis by regulating cholesterol efflux from macrophages. Introduction Clinical and epidemiological studies show a robust, inverse association of HDL cholesterol (HDL-C) levels with cardiovascular disease (CVD) risk (1, 2). Moreover, genetically engineered deficiencies in proteins implicated in HDL metabolism markedly affect atherosclerosis in hypercholesterolemic mice (3, 4), placing HDL in the causal pathway of accelerated atherogenesis in animal models. These observations have trig- Conflict of interest: J. Heinecke is gered intense interest in targeting HDL-C for therapeutic intervention. named as a coinventor on patents from Levels of HDL-C do not necessarily reflect the concentrations of cardioprotective HDL particles, however. the US Patent Office (patents are listed HDL is a collection of macromolecules ranging in size from less than 7 nm to more than 14 nm (5), and the cho- at the end of the supplemental material) lesterol content of the different species can vary 3-fold. Indeed, several lines of evidence suggest that the associa- on the use of HDL markers to predict the risk of cardiovascular disease and has tion between HDL-C levels and CVD status is indirect and that elevating HDL-C is not necessarily therapeutic served as a consultant for Kowa, Merck, (6–12). For example, genetic variations that associate with altered HDL-C do not strongly associate with altered Amgen, Bristol Meyer Squibb, GSK, and CVD risk (9). Trials of drugs such as cholesteryl ester transfer protein inhibitors and niacin, which elevate HDL- Pacific Biomarkers. C by different mechanisms, have failed to reduce events in statin-treated subjects with established CVD (6–8, 10, Submitted: December 9, 2016 11). Moreover, a loss-of-function variant of scavenger receptor B1 (SCARB1) that raises HDL-C levels increases Accepted: June 20, 2017 the risk of CVD in humans (13), and genetic deletion of SCARB1 in mice raises HDL-C levels and promotes Published: August 3, 2017 atherosclerosis (14). These observations indicate that HDL-C does not necessarily reflect HDL’s cardioprotec- tive effects in either humans or mice and that it is critical to examine other functional properties of HDL. Reference information: JCI Insight. 2017;2(15):e92176. HDL’s cardioprotective effect is attributed in part to its ability to mobilize excess cholesterol from https://doi.org/10.1172/jci. artery wall macrophages (15–18). The first key step involves ABCA1 — an ATP-binding cassette trans- insight.92176. porter found in the plasma membrane of macrophages — that exports phospholipids and cholesterol from insight.jci.org https://doi.org/10.1172/jci.insight.92176 1 RESEARCH ARTICLE cells (15–17). Lipid-poor apolipoprotein A-I (APOA1), HDL’s major protein, is thought to be the dominant ligand for ABCA1 in vivo, but recent studies indicate that mature lipidated HDL particles also promote efflux by this pathway (13–15, 17, 19, 20). Mouse and human studies have shown that defects in the APOA1/ABCA1 pathway are important determinants of CVD. For example, ablating the Abca1 gene in myeloid cells increases atherosclerotic lesions in hypercholesterolemic mice (21), while overexpressing human ABCA1 in myeloid cells in hyper- cholesterolemic mice retards atherogenesis (22). Importantly, humans deficient in ABCA1 (Tangier’s dis- ease) accumulate cholesterol-laden macrophages in many different tissues despite having low levels of LDL-cholesterol, providing strong evidence that ABCA1 conducts a key step in sterol excretion from these cells in humans (23). Cholesterol efflux capacity (CEC) — the ability of human serum depleted of lipoproteins containing apolipoprotein B (APOB) to promote cholesterol efflux from macrophages — can vary markedly between different subjects despite similar levels of HDL-C and APOA1 (24). Macrophage CEC of serum HDL associated strongly and negatively with prevalent CVD status in 2 large, independent human popula- tions (25). That association was independent of HDL-C and APOA1 levels. CEC inversely associated with future cardiac events in the Dallas Heart and the EPIC-Norfolk studies (26, 27). These relationships remained highly significant after correction for other classic lipid risk factors for CVD. The assay in the Dallas Heart Study monitored cholesterol efflux capacity specifically by the ABCA1 pathway (28), while the EPIC-Norfolk Study quantified macrophage CEC (24), which involves ABCA1 and multiple other lipid transporters. Taken together, these observations provide strong evidence that macrophage CEC and ABCA1 CEC are clinically relevant metrics of HDL function that are independent of HDL-C. The mechanisms that mediate CEC in APOB-depleted serum are poorly understood. In the current study, we used mice deficient in HDL apolipoproteins implicated in cholesterol efflux from macrophages (15, 16, 29) and analyzed plasma samples biochemically to investigate those proteins’ roles in macrophage CEC and ABCA1 CEC. Our observations indicate that APOA1, apolipoprotein E (APOE), and plasmino- gen (PLG, which is not an apolipoprotein) are key modulators of CEC, and that PLG-dependent ABCA1 CEC is inhibited by lipoprotein(a) [Lp(a)]. These findings could provide the basis for studies of factors that influence CEC — and therefore cardioprotection — in vivo. Results We quantified CEC with both serum HDL (serum depleted of lipoproteins containing APOB) and plas- ma fractionated by high-resolution size-exclusion chromatography (SEC). Because serum HDL con- tains mature HDL as well as partially lipidated and lipid-free forms